Novel solid-phase refolding method for preparation of scFv-immobilized polystyrene plates with high-antigen-binding activity.

In the present study, we demonstrated site-specific immobilization and solid-phase refolding of single-chain Fv antibodies on hydrophilic polystyrene (phi-PS) plates that was mediated by novel polystyrene binding peptides (PS-tags: RIIIRRIRR), which were originally isolated and optimized in previous studies. Three PS-tag-fused scFvs, namely scFv-PS, scFv-(PS), and scFv-PSII, which were over-expressed in the insoluble fraction of Escherichia coli cells were denatured and site-specifically immobilized onto hydrophilic PS plates in the presence of 0.5-4 M urea and 0.1% Tween 20. The antigen-binding activity of the scFvs was efficiently recovered by washing the surface of the plate with PBS that contained 0.1% Tween 20 (PBST). The solid-phase refolding mediated by PS-tag was successfully applied to several scFvs such as mouse anti-CRP antibodies and an anti-RNase antibody, although further investigation of the versatility of scFv-PSII is needed. The maximal density of PS-tag-fused scFvs was increased more than 15-fold compared with a whole monoclonal antibody (mAb) immobilized on Maxisorp and, consequently, the sensitivity of PS-tag-fused scFvs for CRP in a sandwich ELISA was increased 25-fold. Thus, the novel, solid-phase, refolding method mediated by a PS-tag will be very useful for preparation of solid supports coated with recombinant antibody fragments, which can be used in immunoassays and immuno-separation.

Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag).

Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.

High biological activity of a recombinant protein immobilized onto polystyrene.

The adsorption characteristics of glutathione S-transferases (GST) genetically fused with polystyrene (PS)-binding peptides (PS-tags) on PS plates with increase in hydrophilicity were studied to clarify the mechanisms of the specific interaction between the PS-tag-fused protein and PS plates. GST fused with the PS-tag PS19 (RAFIASRRIKRP) preferentially interacted with hydrophilic PS plates, even in the presence of high concentrations of competitors such as Tween 20 and BSA. Both basic and aliphatic amino acids in the PS-tags were involved in the specific interaction of PS-tags with the surface of the hydrophilic PS plate. Genetic fusion of the PS19 variants, PS19-4 (RAIARRIRR) and PS19-6 (RIIIRRIRR), further improved the immobilization yield of GST in the presence of a high concentration of the competitor BSA (50 mg/mL). The PS19-6 peptide specifically interacted with the surfaces of various hydrophilic PS plates, especially in the presence of Tween 20. Higher remaining activity was detected on all of the hydrophilic PS plates immobilized with GST-PS19-6 in comparison with those with wild-type GST and GST-PS19, and the remaining activity was further increased by the addition of Tween 20 in the adsorption state. The PS19-6 peptide developed in this study is therefore very useful as an affinity tag that can immobilize a target protein directly onto various hydrophilic PS supports with high remaining activity.

Efficient immobilization of a ligand antibody with high antigen-binding activity by use of a polystyrene-binding peptide and an intelligent microtiter plate.

A method for immobilization of ligand antibody to improve the efficiency and sensitivity of a sandwich enzyme-linked immunosorbent assay (ELISA) was investigated by the use of anti-TNF-alpha monoclonal antibody chemically conjugated with a polystyrene-binding peptide (PS-tag) and an intelligent microtiter plate with large surface area. We compared both adsorption and antigen-binding activity of the ligand antibody (mAb) and mAb with the PS-tag (mAb-PS-tag) on 3 different PS plates: a hydrophobic PS plate (PS-F-1 plate), a hydrophilic PS plate (PS-A plate), and an intelligent microtiter plate packed with PS beads (PS-E plate). Contact areas of the PS-E plate toward ligand antibody solutions were 7-fold larger than those of conventional PS-F-1 and PS-A plates and consequently, both mAb and mAb-PS-tag were efficiently immobilized on the surface of the PS-E plate due to the significantly enhanced surface area. In particular, when the non-ionic surfactant, Tween20, was present during adsorption of the ligand antibodies, the mAb-PS-tag was site-specifically immobilized on the surface of the PS-E plates and exhibited the highest specific antigen-binding activity. High specific antigen-binding activities were preserved, even though the density of the mAb-PS-tag immobilized on the PS-E plate was reduced with application of smaller volumes. Consequently, the decline in the signal intensities detected from the sandwich ELISA on the PS-E plate was negligible, even when one-fourth the original volume of mAb-PS-tag solution was used to coat the plate. The ligand antibody was immobilized with retention of high antigen-binding activity due to the large surface area of the PS-E plate, specific recognition of the PS-tag, and minimal non-specific interaction of the ligand antibody due to use of Tween 20. Thus, the immobilization method developed in this study resulted in an efficient and highly sensitive sandwich ELISA system that requires smaller amounts of ligand antibodies. This method may be useful for fabrication of protein-based biochips, such as antibody chips.